Different conformations of the German shepherd dog breed affect its posture and movement.

The conformation of the German shepherd dog (GSD) varies considerably within the breed. These differences may result in large variation in the movement and limb loading and undesirable consequences to their musculoskeletal health. This study aimed to investigate the relationship between conformation and biomechanical measures in 60 GSDs. Full body kinematic and kinetic measures were computed from 3D motion capture and pressure data. The dogs were divided into groups based on their back slope and curvature. Correlation analysis and statistical differences between groups showed that GSDs with a greater back slope have a greater contact area in their forelimbs and place them closer together when standing (n = 60). During trot, the dogs with sloped back showed a greater vertical force in the forelimbs and a greater mid-thoracic flexion (n = 60). Unilateral differences were found in the stifle flexion, hock flexion and hock adduction, suggesting greater movement asymmetry with an increase in the back slope (n = 30). In conclusion, several biomechanical parameters are affected by the GSD's slope of the back and not by its curvature. Further studies are required to determine whether the variation in movement, posture and conformation within the breed relates to an increased susceptibility to musculoskeletal disorders.

Allelopathic interference of alfalfa (Medicago sativa L.) genotypes to annual ryegrass (Lolium rigidum).

Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.

A Mathematical Model of Granulopoiesis Incorporating the Negative Feedback Dynamics and Kinetics of G-CSF/Neutrophil Binding and Internalization.

We develop a physiological model of granulopoiesis which includes explicit modelling of the kinetics of the cytokine granulocyte colony-stimulating factor (G-CSF) incorporating both the freely circulating concentration and the concentration of the cytokine bound to mature neutrophils. G-CSF concentrations are used to directly regulate neutrophil production, with the rate of differentiation of stem cells to neutrophil precursors, the effective proliferation rate in mitosis, the maturation time, and the release rate from the mature marrow reservoir into circulation all dependent on the level of G-CSF in the system. The dependence of the maturation time on the cytokine concentration introduces a state-dependent delay into our differential equation model, and we show how this is derived from an age-structured partial differential equation model of the mitosis and maturation and also detail the derivation of the rest of our model. The model and its estimated parameters are shown to successfully predict the neutrophil and G-CSF responses to a variety of treatment scenarios, including the combined administration of chemotherapy and exogenous G-CSF. This concomitant treatment was reproduced without any additional fitting to characterize drug-drug interactions.

Resonant quantum transitions in trapped antihydrogen atoms.

The hydrogen atom is one of the most important and influential model systems in modern physics. Attempts to understand its spectrum are inextricably linked to the early history and development of quantum mechanics. The hydrogen atom's stature lies in its simplicity and in the accuracy with which its spectrum can be measured and compared to theory. Today its spectrum remains a valuable tool for determining the values of fundamental constants and for challenging the limits of modern physics, including the validity of quantum electrodynamics and--by comparison with measurements on its antimatter counterpart, antihydrogen--the validity of CPT (charge conjugation, parity and time reversal) symmetry. Here we report spectroscopy of a pure antimatter atom, demonstrating resonant quantum transitions in antihydrogen. We have manipulated the internal spin state of antihydrogen atoms so as to induce magnetic resonance transitions between hyperfine levels of the positronic ground state. We used resonant microwave radiation to flip the spin of the positron in antihydrogen atoms that were magnetically trapped in the ALPHA apparatus. The spin flip causes trapped anti-atoms to be ejected from the trap. We look for evidence of resonant interaction by comparing the survival rate of trapped atoms irradiated with microwaves on-resonance to that of atoms subjected to microwaves that are off-resonance. In one variant of the experiment, we detect 23 atoms that survive in 110 trapping attempts with microwaves off-resonance (0.21 per attempt), and only two atoms that survive in 103 attempts with microwaves on-resonance (0.02 per attempt). We also describe the direct detection of the annihilation of antihydrogen atoms ejected by the microwaves.

Centrifugal separation and equilibration dynamics in an electron-antiproton plasma.

Charges in cold, multiple-species, non-neutral plasmas separate radially by mass, forming centrifugally separated states. Here, we report the first detailed measurements of such states in an electron-antiproton plasma, and the first observations of the separation dynamics in any centrifugally separated system. While the observed equilibrium states are expected and in agreement with theory, the equilibration time is approximately constant over a wide range of parameters, a surprising and as yet unexplained result. Electron-antiproton plasmas play a crucial role in antihydrogen trapping experiments.

Autoresonant excitation of antiproton plasmas.

We demonstrate controllable excitation of the center-of-mass longitudinal motion of a thermal antiproton plasma using a swept-frequency autoresonant drive. When the plasma is cold, dense, and highly collective in nature, we observe that the entire system behaves as a single-particle nonlinear oscillator, as predicted by a recent theory. In contrast, only a fraction of the antiprotons in a warm plasma can be similarly excited. Antihydrogen was produced and trapped by using this technique to drive antiprotons into a positron plasma, thereby initiating atomic recombination.

Trapped antihydrogen.

Antimatter was first predicted in 1931, by Dirac. Work with high-energy antiparticles is now commonplace, and anti-electrons are used regularly in the medical technique of positron emission tomography scanning. Antihydrogen, the bound state of an antiproton and a positron, has been produced at low energies at CERN (the European Organization for Nuclear Research) since 2002. Antihydrogen is of interest for use in a precision test of nature's fundamental symmetries. The charge conjugation/parity/time reversal (CPT) theorem, a crucial part of the foundation of the standard model of elementary particles and interactions, demands that hydrogen and antihydrogen have the same spectrum. Given the current experimental precision of measurements on the hydrogen atom (about two parts in 10(14) for the frequency of the 1s-to-2s transition), subjecting antihydrogen to rigorous spectroscopic examination would constitute a compelling, model-independent test of CPT. Antihydrogen could also be used to study the gravitational behaviour of antimatter. However, so far experiments have produced antihydrogen that is not confined, precluding detailed study of its structure. Here we demonstrate trapping of antihydrogen atoms. From the interaction of about 10(7) antiprotons and 7 × 10(8) positrons, we observed 38 annihilation events consistent with the controlled release of trapped antihydrogen from our magnetic trap; the measured background is 1.4 ± 1.4 events. This result opens the door to precision measurements on anti-atoms, which can soon be subjected to the same techniques as developed for hydrogen.

Evaporative cooling of antiprotons to cryogenic temperatures.

We report the application of evaporative cooling to clouds of trapped antiprotons, resulting in plasmas with measured temperature as low as 9 K. We have modeled the evaporation process for charged particles using appropriate rate equations. Good agreement between experiment and theory is observed, permitting prediction of cooling efficiency in future experiments. The technique opens up new possibilities for cooling of trapped ions and is of particular interest in antiproton physics, where a precise CPT test on trapped antihydrogen is a long-standing goal.

Antiproton, positron, and electron imaging with a microchannel plate/phosphor detector.

A microchannel plate (MCP)/phosphor screen assembly has been used to destructively measure the radial profile of cold, confined antiprotons, electrons, and positrons in the ALPHA experiment, with the goal of using these trapped particles for antihydrogen creation and confinement. The response of the MCP to low energy (10-200 eV, <1 eV spread) antiproton extractions is compared to that of electrons and positrons.

Chromate reduction by immobilized palladized sulfate-reducing bacteria.

Resting cells of Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans NCIMB 8307 were used for the hydrogenase-mediated reduction of Pd(II) to Pd(0). The resulting hybrid palladium bionanocatalyst (Bio-Pd(0)) was used in the reduction of Cr(VI) to the less environmentally problematic Cr(III) species. The reduction of Cr(VI) by free and agar-immobilized Bio-Pd(0) was evaluated. Investigations using catalyst suspensions showed that Cr(VI) reduction was similar ( approximately 170 nmol Cr(VI)/h/mg Bio-Pd(0)) when Bio-Pd(0) was produced using D. vulgaris or D. desulfuricans. Continuous-flow studies using D. vulgaris Bio-Pd(0) with agar as the immobilization matrix investigated the effect of Bio-Pd(0) loading, inlet Cr(VI) concentration, and flow rate on the efficiency of Cr(VI) reduction. Reduction of Cr(VI) was highest at a D. vulgaris Bio-Pd(0) loading of 7.5 mg Bio-Pd(0)/mL agar (3:1 dry cell wt: Pd(0)), an input [Cr(VI)] of 100 microM, and a flow rate of 1.75 mL/h (approx. 3.5 column volumes/h). A mathematical interpretation predicted the activity of the immobilized Bio-Pd(0) for a given set of conditions within 5% of the value found by experiment. Considering the system as an 'artificial enzyme' analog and application of applied enzyme kinetics gave an apparent K(m) value (K(m app)) of 430 microM Cr(VI) and a determined value of flow-through reactor activity which differed by 11% from that predicted mathematically.

Reduction of Cr(VI) by immobilized cells of Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776.

Hexavalent chromium, a carcinogen and mutagen, can be reduced to Cr(III) by Desulfovibrio vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776. This study examined Cr(VI) reduction by immobilized cells of the two strains in a common solution matrix using various entrapment matrices. Chitosan and PVA-borate beads did not retain integrity and supported low or no reduction of Cr(VI) by the cells. A commercial preparation (Lentikats) was stable but also did not support Cr(VI) reduction. K-carrageenan beads were stable in batch suspensions but gel integrity was lost after only 5 h in a flow-through system in the presence of 100 microM Cr(VI). The best immobilization matrices were agar and agarose, where the initial rates of reduction of Cr(VI) (from 500 microM solution) for D. vulgaris NCIMB 8303 and Microbacterium sp. NCIMB 13776 were 127 (agar) and 130 (agarose), and 15 (agar) and 12 (agarose) nmol h(-1) mg dry cell wt(-1), respectively. The higher removal of Cr(VI) by D. vulgaris was also seen in 14-mL packed-bed flow-through columns, where, at a flow rate of 2.4 mL h(-1), the percentage removal of Cr(VI) was approximately 95% and 60% for D. vulgaris and Microbacterium sp., respectively (agar-immobilized cells). The Cr(VI) reducing activities of D. vulgaris and Microbacterium sp. were lost after 159 and 140 h, respectively. Examination of the beads for structural integrity within the columns in situ using magnetic resonance imaging after 24 and 100 h of continuous operation against Cr(VI) (with negligible Cr retained within the columns) showed that agar beads were more stable with time. The most appropriate system for development of a continuous bioprocess is thus the use of D. vulgaris NCIMB 8303 immobilized in an agar gel matrix.

Applications of bacterial hydrogenases in waste decontamination, manufacture of novel bionanocatalysts and in sustainable energy.

Bacterial hydrogenases have been harnessed to the removal of heavy metals from solution by reduction to less soluble metal species. For Pd(II), its bioreduction results in the deposition of cell-bound Pd(0)-nanoparticles that are ferromagnetic and have a high catalytic activity. Hydrogenases can also be used synthetically in the production of hydrogen from sugary wastes through breakdown of formate produced by fermentation. The Bio-H(2) produced can be used to power an electrical device using a fuel cell to provide clean electricity. Production of hydrogen from confectionery wastes by one organism (Escherichia coli) can be used as the electron donor for the production of Bio-Pd(0) from soluble Pd(II) by a second organism. The resulting Bio-Pd(0) can then be used as a bioinorganic catalyst in the remediation of Cr(VI)-contaminated solutions or polychlorinated biphenyls at the expense of Bio-H(2), as a hydrogenation catalyst for industry or as a component of a fuel cell electrode.

Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris.

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd(0)), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd(0), immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd(0) and 'chemical Pd(0)' were packed into continuous-flow reactors, and challenged with a solution containing 100 microM Cr(VI) (pH 7) at a flow rate of 2.4 ml h(-1). Agar-immobilized chemical Pd(0) columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd(0) maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.

cDNA array analysis of pineal gene expression reveals circadian rhythmicity of the dominant negative helix-loop-helix protein-encoding gene, Id-1.

The pineal gland is a major output of the endogenous vertebrate circadian clock, with melatonin serving as the output signal. In many species, elevated nocturnal melatonin production is associated with changes in pineal gene expression. In the current study, cDNA array analysis was used in an attempt to identify additional genes that exhibit day/night differential expression in the rat pineal gland. This revealed 38 candidate genes, including Id-1 (inhibitor of DNA binding and differentiation). Id-1 encodes a helix-loop-helix (HLH) protein that lacks a basic DNA binding domain and could affect pineal physiology via a dominant negative trans-acting regulatory activity. For this reason Id-1 was selected for further analysis. Id-1 was expressed in a major population of pineal cells and the Id-1 protein was associated with a nuclear complex. The levels of Id-1 mRNA and protein exhibit approximately six-fold day/night rhythms. In contrast, the related genes Id-2 and Id-3 do not exhibit marked day/night differences in pineal expression. Rhythmic Id-1 expression is primarily limited to a C-terminally extended splice variant of Id-1, which would restrict the functional output of the rhythm to protein binding partners of this isoform of Id-1. Our findings add to the body of evidence indicating that transcriptional regulators play a role in neuroendocrine rhythms, and extend this by introducing the concept of a dominant negative HLH involvement. The rhythm in Id-1 in the pineal gland provides an experimental opportunity to identify Id-1-binding partners which may also be involved in Id-1 activity in other functional contexts.

Optimisation of methods for selecting candidate genes from cDNA array screens: application to rat brain punches and pineal.

DNA arrays are potentially powerful experimental tools within neuroscience but application of this technology to in vivo paradigms may, in practice, be limited by the sensitivity of transcript detection and inter-screen variation. Here we describe the use of brain punch micro-sampling, used in combination with commercially available cDNA arrays, for profiling brain gene expression in a mutant strain of rat (GAERS model of absence epilepsy). Furthermore, we describe a multi-step optimisation of analysis methods which provides for improved sensitivity and absence of bias in the selection of candidate genes which may be differentially expressed in the mutant. Our method has been validated through application to a second paradigm, rhythmic gene expression in the rat pineal gland. Our experimental design, and analysis method should therefore be generally applicable to subtle discriminations of transcript abundance within discrete brain areas.

Tissue-specific transgenic knockdown of Fos-related antigen 2 (Fra-2) expression mediated by dominant negative Fra-2.

Fos-related antigen 2 (Fra-2) is a member of the Fos family of immediate-early genes, most of which are rapidly induced by second messengers. All members of this family act by binding to AP-1 sites as heterodimeric complexes with other proteins. However, each appears to have a distinct role. The role and biology of Fra-2 are less well understood than those of its relatives c-Fos, Fra-1, and FosB; moreover, Fra-2 target genes remain largely unknown, as does the basis of its selective effects on transcriptional activity. To pursue these issues, we created a transgenic rat line (NATDNF2) in which a dominant negative fra-2 (DNF2) gene is strongly expressed in the pineal gland; tissue selectivity was achieved by putting the DNF2 gene under the control of the rat arylalkylamine N-acetyltransferase (AANAT) regulatory region, which targets gene expression to a very restricted set of tissues (pineal gland >> retina). Expression of AANAT is normally turned on after the onset of darkness in the rat; as a result, pineal DNF2 expression occurs only at night. This was associated with marked suppression of the nocturnal increase in fra-2 mRNA and protein levels, indicating that DNF2 expression inhibits downstream effects of Fra-2, including the maintenance of high levels of fra-2 gene expression. Analysis of 1,190 genes in the NATDNF2 pineal gland, including the AANAT gene, identified two whose expression is strongly linked to fra-2 expression: the genes encoding type II iodothyronine deiodinase and nectadrin (CD24).

Reversal of metabolic block in glycolysis by enzyme replacement in triosephosphate isomerase-deficient cells.

Inherited deficiency of the housekeeping enzyme triosephosphate isomerase (TPI) is the most severe clinical disorder of glycolysis. Homozygotes manifest congenital hemolytic anemia and progressive neuromuscular impairment, which in most cases pursues an inexorable course with fatal outcome in early childhood. No effective therapy is available. Hitherto specific enzyme replacement has not been attempted in disorders of glycolysis. Primary skeletal muscle myoblasts and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines generated from homozygous TPI-deficient patients were cultured in the presence of exogenous enzyme or cocultured with human K562 erythroleukemia cells as an exogenous source of TPI. Uptake of active enzyme by TPI-deficient cells resulted in reversal of intracellular substrate accumulation, with a reduction in dihydroxyacetone phosphate (DHAP) concentration to levels seen in TPI-competent cells. Evidence of successful metabolic correction of TPI deficiency in vitro establishes the feasibility of enzyme replacement therapy, and has important implications for the potential role of allogeneic bone marrow transplantation and gene therapy as a means of sustained delivery of functional enzyme in vivo.

The consequence of nucleotide substitutions in the triosephosphate isomerase (TPI) gene promoter.

Mutations at -5A-->G, -8-->GA within the cap proximal element (CPE), and -24T-->G within the TATA box of the triosephosphate isomerase (TPI) gene promoter have been identified in populations with a wide geographical distribution. These mutations lie within, or in close proximity to, known cis-active elements in the TPI gene promoter. To determine the functional significance of mutation at these sites, which remains controversial, their effect on the expression of erythrocyte TPI enzyme activity was studied in 110 healthy unrelated subjects. The -5G mutation did not alter erythrocyte TPI level, whereas the -8A mutation was accompanied by a significant reduction in enzyme activity to around 90% and 76% of normal erythrocyte TPI activity in heterozygotes and homozygotes, respectively. The -8A -24G genotype was associated with 75% of normal TPI activity in a heterozygote studied, implying that substitution of G at position -24 within the canonical TATA motif causes an additive decrease in TPI gene transcription in erythroid cells. A DNA-protein complex of 125kDa which was competitively blocked by specific unlabelled oligomers was demonstrated at the CPE and TATA box by electrophoretic mobility shift analysis. These findings provide direct evidence that TPI promoter mutations are linked to a reduction of TPI enzyme activity in vivo.

Ancestral origin of variation in the triosephosphate isomerase gene promoter.

A high frequency of nucleotide substitutions -5A/G, -8G/A, -24T/G in the triosephosphate isomerase (TPI) gene promoter has been demonstrated in African-Americans. The biological significance of these promoter variants, two of which, -8G/A and -24T/G, occur within regulatory elements essential for transcription, is controversial. The geographical distribution and frequency of allelic variation in the TPI promoter was determined in 378 unrelated normal subjects from sub-Saharan African (n = 103), Caribbean (n = 26), Northern European (n = 57), Mediterranean (n = 55), Middle Eastern (n = 42), Asian Indian (n = 48) and Oriental (n = 47) populations. Five haplotypes were identified: the common haplotype, -5A-8G-24T, -5G, -8A, -5G-8A, and -5G-8A-24G. All, with the exception of the -8A haplotype, were present in geographically dispersed populations. The -5G allele, which was found at varying frequency in all groups, has attained high frequency in the African, Caribbean and Oriental populations. Phylogenetic comparison suggests this may represent the ancestral promoter haplotype. Homozygosity for the -5G-8A haplotype identified in four subjects confirms that these variants are not responsible for a null allele as formerly postulated. Linkage disequilibrium between related TPI promoter haplotypes, -5G, -5G-8A and -5G-8A-24G, and a single nucleotide polymorphism at nt2262 of the TPI gene supports a single ancestral origin for these mutations which precedes the separation of African and non-African populations.